In Gram-negative bacteria, lipid A constitutes the hydrophobic membrane anchor of the unique glycolipid lipopolysaccharide (LPS). It is essential for viability and serves as a potent immunostimulant by activating the TLR4 receptor. The structure and biosynthetic pathway of Escherichia coli lipid A has been extensively haracterized over the years. It is a hexaacylated disaccharide of glucosamine that is substituted at the 1- and 4'-positions with phosphate. Recently, the lipid A structure of the spirochete Leptospira interrogans has been solved, and two interesting features were revealed. The 1-phosphate is methylated, and the 4'-position is dephosphorylated. It has also been discovered that leptospiral LPS activates TLR2 in the innate immune response instead of TLR4. Understanding the enzymology of these modifications will not only facilitate the understanding of lipid A biosynthesis, but it will also provide new molecular insights into pathogenesis. Specifically, this proposal focuses on (l-ll) identification and characterization of the 1-methyltransferase and pie 4'-phosphatase; and (III) investigation of the effects these structural modifications have on the unique rTLR2-dependent immune response.